The overall goal of this program project is to identify the cellular and molecular triggers that initiate fatal cardiac arrhythmias and to determine novel molecular-targeted therapeutic strategies to treat them. The central hypothesis is that sudden cardiac death (SCD) can be caused by disruption of molecular complexes and processes that, in normal hearts, underlie balanced regulation of cellular activity and furthermore that altered cellular calcium (Ca) homeostasis plays a critical role in triggering the resulting arrhythmic activity. It is thus the fundamental assumption of this program that, to understand the mechanistic basis of the events that underlie SCD, an integrative approach is necessary that considers variants of ion channel molecular complexes that coordinate the function of intracellular (Project 1) and surface (sarcolemmal) (Project 2) ion channels as well as a critical and central role played by intracellular Ca regulation (Project 3). A key aspect of this Program is the Animal Core B that will maintain and provide genetic mouse models to each of the three PIs. This project (Project 1) focuses on elucidating defects in the regulation of type 2 ryanodine receptor (RyR2)/SR Ca release channel, which is required for cardiac excitation-contraction (EC) coupling. We propose to test the hypothesis that a diastolic SR Ca "leak" via defective RyR2 can serve as triggers for fatal cardiac arrhythmias which cause sudden cardiac death (SCD). A new theme that has emerged from this project is the concept that there are genes that encode proteins which are "protective" against SCD. During the previous funding period of this project the applicant identified a defect in RyR2 in patients with exercise-induced SCD: "leaky" RyR2 channels that exhibit decreased binding affinity for the stabilizing subunit calstabin2 (FKBP12.6). This observation has directly lead to the development of a novel potential therapy for SCD, RyCal (JTV-S36, which is a JTV-519 derivative without HERG and L-type channel blocking activities), representing a new class of intracellular calcium channel stabilizers that prevent exercise-induced SCD in mouse models. During the previous funding period the applicant identified the phosphodiesterase PDE4D3 as a novel component of the RyR2 macromolecular signaling complex and preliminary data, presented in this proposal, suggest that PDE4D3 in the RyR2 complex may be "protective" against SCD because mice that are deficient in PDE4D3 are predisposed to exercise-induced SCD. Moreover, a mouse engineered to express an RyR2 channel that cannot be PKA phosphorylated, RyR2-S2808A, is protected against SCD induced by inhibition of PDE4 with rolipram. Close interactions with the PIs of Project 2 (R. Kass - voltage-gated ion channels) and Project 3 (W.J. Lederer - calcium signaling) enhances each of the proposed aims as detailed below. Thus this work has the potential to determine a mechanistic basis for Sudden Cardiac Death (SCD) at the molecular level, and to develop therapeutic strategies in man based on specific molecular and mechanistic models.